Skeletal muscle release of glutamine and alanine is thought to provide substrate for metabolic events in other tissues during early fasting and hypercatabolic states. However, the fate of the nitrogen and of the carbon skeletons of these amino acids after being extracted by other organs (notably the liver, gut, and kidney) is not well understood. For example, the so-called glucose-alanine cycle accounts for the interorgan transport of alanine C while concomitant transamination of muscle branched-chain amino acids (BCAA's) provides an important alanine N source. However, BCAA N contribution to glutamine N, the major carrier of N released from muscle, is unknown in the intact organism in vivo. Part of the problem in answering these questions has been lack of analytical methodology for tracing the flow of N in the body. This project proposes to develop new analytical methodology for measuring glutamate and glutamine amino-15N enrichments and new hybrid GCMS instrumentation for measuring the transfer of amino acid tracer 15N enrichment to other amino acids and N end-products. These methods will be used to investigate BCAA, glutamate, glutamine, and alanine metabolism in man and interorgan transport in the dog using stable isotope amino acid tracers. The proposed studies are designed to extend our previous investigations of the mechanism of BCAA metabolic control, to define the contribution of the BCAA N to glutamine and alanine formation, to define the fate of glutamine and alanine N, and to study the regulation of glutamate/glutamine and alanine metabolism as a function of dietary protein intake.